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U.S. Department of Health and Human Services

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M-I-05-3: 2400h-3 ELECTRONIC SOMATIC CELL COUNT Fossomatic 5000

June 2, 2005
 

Milk Safety References
National Conference on Interstate Milk Shipments (NCIMS)

[Unless otherwise stated all tolerances are ±5%]

Sample
  1. __________ Laboratory Requirements (see CP, item 33 & 34)
    1. __________ Un-preserved samples may be run up to 72 hours after initial collection
    2. __________ Samples may be run up to 7 days after initial collection if preserved with 0.02% 2-bromo-2-nitropropane-1,3-diol (BronopolTM) or 0.05% potassium dichromate (K2Cr2O7)
    3. __________ Comparative test with DMSCC
      1. __________ Performed by each analyst performing ESCC test
      2. __________ Test 4 samples (100K-200K, 300K-500K, 600K-800K and 900K-1.2M) in triplicate for both DMSCC (three separate smears each) and ESCC (three separate sub-samples each, do not read same sample three times)
      3. __________ Results must be shown to be acceptable prior to official testing by analyst performing comparison, i.e. analyst is not certified until found acceptable. (co-requisite for certification)
      4. __________ Copy of comparison and results in QC record (or easily accessible file in laboratory)
    4. __________ Analysts certified in DMSCC
    APPARATUS
  2. __________ See Cultural Procedures, items 1-5
  3. __________ Water Bath
    1. __________ Circulating and thermostatically controlled to 37-42C
    REAGENTS
  4. __________ Preparation
    1. __________ Fixed volume or electronic, 100 µL
      1. __________ Stock Solution: Dissolve 500 mL of Clean 5000 in 4.5 L of deionized (DI) or MS water, heat to about 60C, store in airtight, light proof container in a cool location and use within 16 weeks

        __________ Date Prep. ________ Exp. Date ________

      2. __________ Buffer/diluent Solution: Mix 1 L of stock solution with one bag (354 g) of Buffer 5000, add DI or MS water to 10 L, heat to 40 - 60C to speed process, store in diluent container next to instrument and use within 3 weeks (enough for about 25,000 samples)

        __________ Date Prep. ________ Exp. Date ________

      3. __________ Rinse/sheath Liquid: Mix 250 mL of stock solution with DI or MS water to make 50 L, store and use within 3 weeks (enough for about 5000 samples)

        __________ Date Prep. ________ Exp. Date ________

      4. __________ Insert Dye 5000 bag according to manufacturer's instructions
    2. __________ 0.5 Liter Bags
      1. __________ Stock Solution: Dissolve 100 mL of Clean 5000 in 900 mL of DI or MS water (use syringe), heat to about 60C, store in airtight, light proof container in a cool location and use within 16 weeks

        __________ Date Prep. ________ Exp. Date ________

      2. __________ Buffer/diluent Solution: Mix 0.5 L of stock solution with one bag (171 g) of Buffer 5000, add DI or MS water to 5 L, heat to 40 - 60C to speed process, store in diluent container next to instrument and use within 3 weeks(enough for about 10,000 samples)

        __________ Date Prep. ________ Exp. Date ________

      3. __________ Rinse/sheath Liquid: Mix 100 mL of stock solution with DI or MS water to 20 L, store and use within 3 weeks (enough for about 2000 samples)

        __________ Date Prep. ________ Exp. Date ________

      4. __________ Insert Dye 5000 bag according to manufacturer's instructions
    3. __________ Stock Solutions for 5000 basic
      1. __________ Dye/buffer stock solution: Dissolve 3 ethidium bromide tablets in 1 L of DI or MS water, stir to completely dissolve tablets, store in dark cool location, use within 16 weeks

        __________ Date Prep. ________ Exp. Date ________

      2. __________ Clean 5000 stock solution: Dilute one bottle of Clean 5000 in 4.5 L of DI or MS water, heat to about 40 - 60C to speed process, store and use within 16 weeks

        __________ Date Prep. ________ Exp. Date ________

    4. __________ Working Solutions for 5000 basic
      1. __________ Buffer/diluent solution: Dissolve one bag of Buffer 5000 in about 8 L of DI or MS water in 10 L container, add 1 L of Clean 5000 stock solution and fill to a total of 10 L with DI or MS water, store and use within 6 weeks

        __________ Date Prep. ________ Exp. Date ________

      2. __________ Dye/buffer solution: Mix 1800 mL of Buffer/diluent solution and 200 mL of Dye stock solution in the reagent bottle and place in the instruments according to instructions, use within 6 weeks

        __________ Date Prep. ________ Exp. Date ________

      3. __________ Rinse/sheath liquid: Fill a 50 L container with about 49 L of DI or MS water, add 250 mL of Clean 5000 stock solution and fill to 50 L with deionize water, use within 3 weeks

        __________ Date Prep. ________ Exp. Date ________

  5. __________ Storage Periods and Temperatures
    1. __________ Storage time for Buffer 5000 and Clean 500 is one year from FE (Foss Electric, i.e., manufacture) date

      __________ FE Date ________ Exp. Date ________

    2. __________ Store Dye 5000 at 2-8C for a maximum of 1 year from FE date

      __________ FE Date ________ Exp. Date ________

    3. __________ Rinse 5000, used to rinse dye interface when instrument has not been used for more than 7 days, store for 1 year from FE date, if seal is not broken, after seal is broken store at 2-8C for no more than 6 months

      __________ FE Date ________ Exp. Date ________

  6. __________ Other Liquids
    1. __________ Blind solution: Prepare a 1% (w/v) NaCl solution
    2. __________ Cleaning solution for milk waste tube: Prepare a 0.5% S-470 solution (5 g per Liter), heat to 50 - 70C, used to clean the milk waste tube at end of day, use within 1 week
      Date prep. ________ Exp. Date
    3. __________ Cleaning solution for flow cell flush: Use 0.5% S-470 solution (see item b above)
  7. __________ All solutions labeled with date prepared and expiration date

    START UP

  8. __________ Cell Counter
    1. __________ Turn power on and place instrument in standby mode
    2. __________ Check that enough rinse/sheath liquid, dye and buffer solutions are available in the external containers
    3. __________ Solutions not used beyond expiration date(s)
    4. __________ Perform a blind check before starting measurement, if mean count is < 3000 cells/mL and individual measurements < 5000, within acceptable limits
    5. __________ IF ANY ABOVE PARAMETERS ARE WRONG, CORRECT BEFORE PROCEEDING
    6. __________ Records maintained on all parameters each time instrument is used
  9. __________ Milk Standards
    1. __________ Commercially prepared: ___________________
      Lot# ______ Date Rcd. ______________
      1. __________ Stock Solution: Dissolve 500 mL of Clean 5000 in 4.5 L of deionized (DI) or MS water, heat to about 60C, store in airtight, light proof container in a cool location and use within 16 weeks
      2. __________ Do DMSCC in triplicate on each standard in set and average counts, records maintained
      3. __________ DMSCC check performed in rotation by all certified analysts
      4. __________ Standards used within one week
    2. __________ Certified provider: ___________________
      Lot# ______ Exp. Date ______ Date Rcd. ______
      1. __________ Four standards in ranges 100K-200K, 300K-500K, 600K- 800K and 900K-1.2M
      2. __________ Maintain copies of all provided DMSCC values
      3. __________ Measure and maintain records of temperature (0-7.2C) of standards as received
      4. __________ Maintain copies of all correspondence regarding problems
      5. __________ Standards used by manufacturer's expiration date
    3. __________ Laboratory prepared (weekly)
      1. __________ Prepare from raw milk > 18 hours old preserved with 0.05% potassium dichromate (K2Cr2O7)
      2. __________ Or, preserved with 0.02% 2-bromo-2-nitropropane- 1,3-diol (Bronopol™)
      3. __________ Standards cannot be preserved with formalin
      4. __________ Prepare 4 standards in ranges 100K 200K, 300K 500K, 600K-800K and 900K 1.2M, used within one week

        __________ Date prep. ________ Exp. Date _________

      5. __________ Do DMSCC in triplicate on each standard and average counts, records maintained
      6. __________ DMSCC check performed in rotation by all certified analysts
    4. __________ Hourly Control Sample (instrument drift check)
      1. __________ Use one of the standards (items 9a or b) in the 500- 800K range, run in triplicate and determine average
      2. __________ Optionally, prepare sufficient control/sample 500- 800K range, run in triplicate and determine average
    PROCEDURE
  10. __________ Handling and storage
    1. __________ Heat standards to 37-42C (using a temperature control) and read within 30 minutes of reaching temperature, used once and then discarded, i.e. do not re-use
    2. __________ Mix by inverting at least 2x, place in rack and put onto automatic track, run within 10 minutes
    3. __________ Run the standards three times and average the counts for each level, records maintained
    4. __________ Each standard's average must be within 10% of the DMSCC (item 9) for that level, except within 15% for 100K-200K standard, records maintained
    5. __________ Repeatability - a standard in the 300K to 800K range must have a coefficient of variation (CV) of 5% or less on 10 replicates (Refer to Operating Manual), records maintained
    6. __________ THESE PARAMETERS MUST BE ACHIEVED BEFORE PROCEEDING
    CONTROLS
  11. __________ Testing Samples
    1. __________ Heat samples to 37-42C (using a temperature control) and read within 30 minutes of reaching temperature; samples must not be re-used and must be discarded after use
    2. __________ Mix by inverting at least 2x, place in rack and put onto automatic track, run within 10 minutes of reaching the testing temperature
  12. __________ With continuous operation:
    1. __________ Run a standard or optionally a control/sample (item 9d) in the 500K to 800K range hourly, must be within 5% of the original established instrument average value (optionally, within 10% of original DMSCC average)
    2. __________ Run control 3x
    3. __________ Run zero control (item 8d)
    4. __________ Maintain records
  13. __________ Routine maintenance
    1. __________ Perform as described in operating manual
    2. __________ Maintain records
    REPORTS
  14. __________ Computing and Reporting Counts
    1. __________ Count obtained x 1000 is the cell count/mL milk
    2. __________ In reporting electronic somatic cell counts (ESCC/mL), record only first two left hand digits, raising second digit to next higher number when third digit is 6 or more
    3. __________ Report the two left hand digits (rounded)
      1. __________ If the third digit is 5 the second digit is rounded by the following rule
        1. __________ When the second digit is odd round up, raise the second digit by 1 (odd up, 235 to 240)
        2. __________ When the second digit is even round down, delete the 5 and report the second digit as is (even down, 225 to 220)
    4. __________ If count on instrument is < 100 report as < 100,000 ESCC/ mL